Submitted to: Ecdysone Workshop Proceedings
Publication Type: Abstract Only
Publication Acceptance Date: 5/16/2006
Publication Date: 7/10/2006
Citation: Tavva, V.S., Palli, S.R., Dinkins, R.D., Collins, G.B. 2006. Ecr gene switches for regulated expression of transgenes in plants. Ecdysone Workshop Proceedings. 10-14 July 2006.
Technical Abstract: Genetic engineering of plants through transgene technology is being used to enhance agronomic performance or improve quality traits in a wide variety of plant species, and has become a fundamental tool for basic research in plant biotechnology. Constitutive promoters are presently the primary means used to express transgenes in plants. Metabolic energy waste, negative pleiotropic effects and potential gene escape are some of the disadvantages associated with the use of constitutive promoters. Inducible gene regulation systems based on specific chemicals have many potential applications in agriculture and in the basic understanding of gene function. As a result several gene switches have been developed. However, the properties of the chemicals used in these switches make their use limited to research purposes. An ecdysone receptor gene switch is one of the best inducible gene regulation systems available, because the chemical, methoxyfenozide required for its regulation is registered for field use. An EcR gene switch with a potential for use in large-scale field applications and its applicability to a variety of plant species has been developed by adopting a two-hybrid format. In a two-hybrid switch format, the GAL4 DNA binding domain (GAL4 DBD) was fused to the ligand binding domain (LBD) of the Choristoneura fumiferana ecdysone receptor (CfEcR); and, the VP16 activation domain (VP16 AD) was fused to LBD of Locust migratoria retinoid x receptor (LmRXR) or Homo sapiens retinoid x receptor (HsRXR). Upon application of methoxyfenozide, the heterodimer of these two fusion proteins transactivates the luciferase reporter gene placed under the control of multiple copies of cis acting elements and a minimal 35S promoter. The sensitivity of the CfEcR gene switch was improved from micromolar to nanomolar concentrations of ligand by using the CfEcR:LmRXR two-hybrid combination and a reduction in the background expression levels was achieved by using the CfEcR:HsRXR two-hybrid combination. The performance of EcR gene switch was improved further using Hs-LmRXR chimeras and/or CfEcR mutants. The efficiency of EcR gene switches in inducing the target gene expression was also tested in functional genomic studies by regulating the expression of a Superman-like single zinc finger protein 11 (ZFP11) gene in both Arabidopsis and tobacco plants. In addition, determination of pleiotropic effects of switch components and ligands is also a prerequisite for wide-spread use of these gene regulation systems in research and field applications. Therefore, we have also carried out the microarray analysis of the gene switch Arabidopsis plants to determine if there are pleiotropic effects caused by the introduction of a methoxyfenozide-inducible ecdysone receptor-based gene regulation system. The development of a highly sensitive and tightly regulated EcR gene switch along with other desirable properties such as availability of safe and field registered ligand should provide widespread use for this system.